Andreas Springer

A mannan binding lectin is involved in cell–cell attachment in a toxic strain of Microcystis aeruginosa

Microcystin, a hepatotoxin that represents a serious health risk for humans and livestock, is produced by the bloom‐forming cyanobacterium Microcystis aeruginosa in freshwater bodies worldwide. Here we describe the discovery of a lectin, microvirin (MVN), in M. aeruginosa PCC7806 that shares 33% identity with the potent anti‐HIV protein cyanovirin‐N from Nostoc ellipsosporum. Carbohydrate microarrays were employed to demonstrate the high specificity of the protein for high‐mannose structures containing α(1→2) linked mannose residues. Lectin binding analyses and phenotypic characterizations of MVN‐deficient mutants suggest that MVN is involved in cell–cell recognition and cell–cell attachment of Microcystis. A binding partner of MVN was identified in the lipopolysaccharide fraction of M. aeruginosa PCC7806. MVN is differentially expressed in mutants lacking the hepatotoxin microcystin. Additionally, MVN‐deficient mutants contain much lower amounts of microcystin than the wild‐type cells. We discuss a possible functional correlation between microcystin and the lectin and possible implications on Microcystis morphotype formation. This study provides the first experimental evidence that microcystins may have an impact on Microcystis colony formation that is highly important for the competitive advantage of Microcystis over other phytoplankton species.

Dynamic Motion in Crown Ether Dendrimer Complexes: A “Spacewalk” on the Molecular Scale

Brownian motion, the rotation of molecules, and vibrations within molecules are typical forms of thermal motion. Fast chemical equilibria, such as the inversion at the ammonia nitrogen atom, the interconversion of conformers in alkanes, or highly dynamic association/dissociation processes in weakly bound noncovalent complexes are also thermally induced. In the context of noncovalent complexes, it is fascinating to examine whether an intracomplex migration of a guest molecule between different binding sites of a multitopic host is possible and how a motion like this could be monitored. Herein, the first five generations (G1–G5) of polyamino propylene amine (POPAM) dendrimers serve as prototypical multitopic hosts. We address the question, whether crown ethers can directly move from binding site to binding site on the dendrimers periphery without intermediate dissociation/reassociation (Figure 1). Furthermore, if this molecular “spacewalk” is indeed possible, it raises the question as to by what mechanism it proceeds. In solution, the detection of such an intracomplex binding-site hopping is challenging if not impossible, because it is always superimposed by dissociation/reassociation equilibria. Therefore, it is necessary to isolate the complexes from each other and from the corresponding free building blocks to suppress any intercomplex guest-exchange reactions. The high vacuum inside a mass spectrometer is ideally suited to achieve the isolation of the complexes as the complexes there are like-charged and thus efficiently separated from each other by charge repulsion. Also, reactions with neutral crown ether molecules can be excluded. Fragmentation of the crown ether/dendrimer complexes would be the only source for the appearance of neutral crown ethers in the gas phase. Therefore, their partial pressure is much too low to result in an efficient reattachment during the short time they spend inside the instrument before being pumped away. However, this approach comes with the difficulty that any intramolecular process does not change the complex ion s molecular mass and thus remains undetectable by a simple determination of the mass-to-charge ratio (m/z). Therefore, a gas-phase reaction is required that probes the guest s motion. Such a reaction must a) proceed energetically below the complex dissociation energy, b) cause a mass shift, and c) be directly linked to the guest movement. To realize this idea, we chose POPAM dendrimers as the multitopic scaffold. These dendrimers have highly branched onion-layer-type structures (Figure 1). From each generation (Gn) to the next, the number of peripheral amino groups doubles from four in theG1 dendrimer to 64 inG5. Their gasphase chemistry has been studied in detail. In the absence of a solvating agent, protonation is likely to occur at interior tertiary amines rather than the peripheral primary NH2 groups. To examine the host–guest chemistry of dendritic molecules in the gas phase is generally a challenging and byand-large unexplored field of research. Only a few examples exist to date. In our study, [18]crown-6 serves as the guest, it binds to primary ammonium ions in solution, and in the gas phase. Dendritic crown ether/ammonium complexes are Figure 1. Chemical structure of [18]crown-6 and a fourth generation (G4) POPAM dendrimer. Starting with a 1,4-diaminobutane core, the nth shell of branches is divergently grown on the (n 1)th generation dendrimer by two Michael additions of acrylnitrile to each branch and subsequent hydrogenolytic reduction of the nitrile groups. The red arrows symbolize the main question of the present study: Can crown ethers move freely along the periphery of POPAM dendrimers without intermediate dissociation of the complex? As this process proceeds in the high vacuum inside a mass spectrometer, we refer to it as a molecular “spacewalk”.

Evaluation of multivalency as an organization principle for the efficient synthesis of doubly and triply threaded amide rotaxanes

Mono-, di- and trivalent pseudorotaxanes with tetralactam macrocycle hosts and axles containing diamide binding stations as the guests have been synthesised. Their threading behaviour was analyzed in detail by NMR experiments and isothermal titration calorimetry. An X-ray crystal structure of the monovalent pseudorotaxane confirms the binding motif. Double mutant cycle analysis provides the effective molarities and insight into the chelate cooperativity of multivalent binding. While the second binding event in a trivalent pseudorotaxane exhibits a slightly positive cooperativity, the third binding is nearly non-cooperative. Nevertheless, the enhanced binding affinities resulting from the multivalent interaction are the basis for a highly efficient synthesis of di- and trivalent rotaxanes through stoppering the axle termini by “click” chemistry. Evidence for the multiply threaded geometry comes from NMR spectroscopy as well as tandem mass-spectrometric fragmentation experiments of mass-selected rotaxane ions in the gas phase. Furthermore, the trivalent rotaxane can be controlled by external stimuli (chloride addition and removal) which lead to an elevator-type movement of the wheel along the axle.

Gas-phase organocatalysis with crown ethers

Catalytic cycles can be investigated in detail in the gas phase in a step-by-step manner by tandem mass spectrometric experiments that allow for the mass-selection of each of the intermediates prior to the subsequent step. In the present article, we describe large crown ethers (dibenzo-24-crown-8 to dibenzo-30-crown-10) to mediate the E2-elimination of propene from crown ether-propylammonium complexes giving rise to NH4+/crown ether complexes. A back exchange of the ammonia against propyl amine completes the catalytic cycle. To the best of our knowledge, this reaction cycle represents the first example of organocatalysis in the gas-phase. In addition, the larger crown ethers significantly accelerate the H/D-exchange reaction of the five NH hydrogen atoms in singly protonated ethylenediamine as compared to the analogous smaller crown ether complexes. This effect, which is not observed for the propylammonium complexes, can be rationalized by a favorable pre-organization of the protonated ethylenediamine ion within the cavity of the crown ether through hydrogen bonding to both the ammonium and the amine groups. Within this complex, the H/D-exchange can proceed through an efficient “relay” mechanism. A comparison of the H/D-exchange behavior of ammonium complexes with differently sized crown ethers reveals a distinct reactivity pattern that strongly depends on the crown ether size. While 18-crown-6 acts as a non-covalent protective group suppressing the H/D-exchange almost completely, smaller and larger crown ethers exhibit higher exchange rates.

Mass spectrometric study of oligourea macrocycles and their anion binding behavior

Two series, one of tris-urea macrocycles and another of hexakis-urea macrocycles, are examined by (tandem) Fourier-transform ion cyclotron resonance (FTICR) mass spectrometry with respect to their fragmentation patterns and anion binding properties. All macrocycles are based on two different building blocks, one of which is a very rigid xanthene unit and the other one is a more flexible diphenyl ether. The composition and the sequence of these units thus determine their flexibility. During the fragmentation of deprotonated oligourea macrocycles in the gas phase, one urea N-CO bond is cleaved followed by a scrambling reaction within the macrocycle structure. Consequently, fragments are observed that deviate from those that would be expected from the sequence of the subunits. Interesting anion binding properties involve the simultaneous recognition of two chloride anions by one of the hexakis-urea macrocycles, whose flexibility allows this host to form a double-helical structure. Flexibility also determines which of the hexameric receptors bears a high sulfate affinity. The interaction energy between some of the macrocycles and sulfate is high enough to even stabilize the intrinsically unstable sulfate dianion.

Comparability study of Rituximab originator and follow-on biopharmaceutical

&NA; Immunglobolin G (IgG)‐based biopharmaceuticals are emerging on the pharmaceuticals market due to their high target selectivity in different diseases. In parallel, a growing interest by other companies to produce similar or highly similar follow‐on biologics exits, once the patent of blockbuster biotherapeutics is about to expire. In correlation to their complex structure, an analytical challenge is facing the approval of these biosimilars. Health authorities (e.g. FDA and EMA) have issued several guidelines to define critical quality attributes during manufacturing process changes. In the current study, physicochemical characterization using state‐of‐the‐art analytics was applied to analyse intact mass, post‐translational modifications (PTMs) and higher order structure of Rituximab and one of its biosimilars. Intact mass analysis, middle‐up approach as well as subunit analysis revealed similar glycoforms but additional lysine variants in the biosimilar. The N‐glycosylation site was confirmed for both, the originator and the biosimilar. PTMs and higher order structure were confirmed to be similar. A special focus was given to N‐glycosylation due to its potential to monitor the batch‐to‐batch consistency and alteration during the production bioprocess. Comparison of the N‐glycosylation profiles obtained from three batches of the biosimilar and the reference product showed quantitative variations, although the N‐glycans were qualitatively similar. Furthermore, a head‐to‐head comparability of functional properties was performed to investigate the impact of glycosylation alteration and PTMs on potency within the biosimilar batches and between originator and follow‐on biodrug. The data affirm that the difference is still in the acceptable range for biosimilarity. HighlightsMass spectrometry‐based methods to investigate intact mass, PTMs and higher order structure of biopharmaceuticals.Establishment of a comparability study between candidate originator and follow‐on biopharmaceutical.Monitoring of batch‐to‐batch changes in glycosylation as a critical quality attribute (CQA).Functional properties of three biopharmaceutical batches to investigate the impact of glycosylation on the potency.

Economical, Plain, and Rapid Authentication of Actaea racemosa L. (syn. Cimicifuga racemosa, Black Cohosh) Herbal Raw Material by Resilient RP‐PDA‐HPLC and Chemometric Analysis

INTRODUCTION The medicinal plant Actaea racemosa L. (Ranunculaceae, aka black cohosh) is widely used to treat climacteric complaints as an alternative to hormone substitution. Recent trials prove efficacy and safety of the approved herbal medicinal products from extracts of pharmaceutical quality. This led to worldwide increasing sales. A higher demand for the plant material results in problems with economically motivated adulteration. Thus, reliable tools for herbal drug authentication are necessary. OBJECTIVE To develop an economical, plain, and rapid method to distinguish between closely related American and Asian Actaea species, using securely established and resilient analytical methods coupled to a chemometric evaluation of the resulting data. METHODOLOGY We developed and validated a RP-PDA-HPLC method including an extraction by ultra-sonication to determine the genuine contents of partly hydrolysis-sensitive polyphenols in Actaea racemosa roots and rhizomes, and applied it to a large number of 203 Actaea samples consisting of seven species. RESULTS We were able to generate reliable data with regards to the polyphenolic esters in the samples. The evaluation of this data by principle component analysis (PCA) made a discrimination between Asian Actaea species (sheng ma), one American Actaea species (Appalachian bugbane), and A. racemosa possible. CONCLUSION The developed RP-PDA-HPLC method coupled to PCA is an excellent tool for authentication of the Actaea racemosa herbal drug, and can be a powerful addition to the TLC methods used in the dedicated pharmacopoeias, and is a promising alternative to expensive and lots of expertise requiring methods. Copyright

A novel preventive therapy for paclitaxel-induced cognitive deficits: preclinical evidence from C57BL/6 mice

Chemotherapy-induced central nervous system (CNS) neurotoxicity presents an unmet medical need. Patients often report a cognitive decline in temporal correlation to chemotherapy, particularly for hippocampus-dependent verbal and visuo-spatial abilities. We treated adult C57Bl/6 mice with 12 × 20 mg kg−1 paclitaxel (PTX), mimicking clinical conditions of dose-dense chemotherapy, followed by a pulse of bromodesoxyuridine (BrdU) to label dividing cells. In this model, mice developed visuo-spatial memory impairments, and we measured peak PTX concentrations in the hippocampus of 230 nm l−1, which was sevenfold higher compared with the neocortex. Histologic analysis revealed a reduced hippocampal cell proliferation. In vitro, we observed severe toxicity in slowly proliferating neural stem cells (NSC) as well as human neuronal progenitor cells after 2 h exposure to low nanomolar concentrations of PTX. In comparison, mature post-mitotic hippocampal neurons and cell lines of malignant cells were less vulnerable. In PTX-treated NSC, we observed an increase of intracellular calcium levels, as well as an increased activity of calpain- and caspase 3/7, suggesting a calcium-dependent mechanism. This cell death pathway could be specifically inhibited with lithium, but not glycogen synthase kinase 3 inhibitors, which protected NSC in vitro. In vivo, preemptive treatment of mice with lithium prevented PTX-induced memory deficits and abnormal adult hippocampal neurogenesis. In summary, we identified a molecular pathomechanism, which invokes PTX-induced cytotoxicity in NSC independent of cell cycle status. This pathway could be pharmacologically inhibited with lithium without impairing paclitaxel’s tubulin-dependent cytostatic mode of action, enabling a potential translational clinical approach.

NIR Spectroscopy of Actaea racemosa L. rhizome – En Route to Fast and Low-Cost Quality Assessment

Rhizomes of Actaea racemosa L. (formerly Cimicifuga racemosa) gained increasing interest as a plant-derived drug due to its hormone-like activity and the absence of estrogenic activity. According to the Current Good Manufacturing Practices guidelines and pharmacopeial standards, quality assessment of herbal starting materials includes tests on identity and substitution, as well as quantification of secondary metabolites, usually by HPTLC and LC methods. To reduce the laboratory effort, we investigated near-infrared spectroscopy for rapid species authentication and quantification of metabolites of interest.Near-infrared spectroscopy analysis is carried out directly on the milled raw plant material. Spectra were correlated with reference data of polyphenols and triterpene glycosides determined by LC/diode array detection and LC/evaporative light scattering detection, respectively. Quantification models were built and validated by cross-validation procedures. Clone plants, derived by vegetative propagation, and plants of a collection from different geographical origins cultivated in Berlin were analysed together with mixed batches from wild harvests purchased at wholesalers.Generally, good to excellent correlations were found for the overall content of polyphenols with coefficients of determination of R2 > 0.93. For individual polyphenols such as fukinolic acid, only models containing clone plants succeeded (R2 > 0.92). For the total content of triterpene glycosides, results were generally worse in comparison to polyphenols and were observed only for the mixed batches (R2 = 0.93).Next to quantitative analysis, near-infrared spectroscopy was proven as a rapid alternative to other, more laborious methods for species authentication. Near-infrared spectroscopy was able to distinguish different Actaea spp. such as the North American Actaea cordifolia and the Asian Actaea cimicifuga, Actaea dahurica, Actaea heracleifolia, and Actaea simplex.

Electrospray ionization mass spectrometric study of mercury complexes of N-heterocyclic carbenes derived from 1,2,4-triazolium salt precursors

By mixing 1,2,4-triazolium salts (precursors of N-heterocyclic carbenes 1–6) with mercury acetate, a number of complexes have been obtained under electrospray ionization condition. Carbenes 1 and 2 contain one carbene center; therefore, they are able to bond only one mercury cation. Carbenes 3–5 contain two carbene centers; therefore, they can bond two mercury cations. Mercury complexes of 1–5 always contain an acetate anion attached to a mercury cation. Carbene 6 also contains two carbene centers; however, its structure allows formation of a complex containing mercury bonded simultaneously to both centers, therefore, the complex that does not contain an acetate anion. The MS/MS spectra taken for complexes of carbenes 1–5 have shown formation of a cation corresponding to N1 substituent (adamantyl or benzyl), and those of complexes of carbenes 3–5 (doubly charged ions) have also shown the respective complementary partner ions. Mercury complex of 2 has yielded some other interesting fragmentation pathways, e.g. a loss of the HHgOOCCH3 molecule. The fragmentation pathway of the mercury complexes of 6 was found to be complicated.